The wells of a Nunc Maxisorp microplate (460−518) were coated with an anti-human Fc goat polyclonal antibody (10 μg/mL) (Jackson ImmunoResearch, West Grove, PA, USA) at 4 °C overnight and then blocked with 0.5% BSA in PBS at room temperature for 2 h. Fc-fused proteins (1 μg/mL) were captured by the antibody, and phage solution (1.0 × 1011 pfu/mL) or biotinylated peptide solution was added to the wells and incubated at room temperature for 1 h. After three times washing with PBST, bound phages or peptides were detected using horseradish peroxidase (HRP)-conjugated anti-T7 antibody (5000-fold dilution in PBS with 0.5% BSA) (Merck Millipore) and HRP-conjugated streptavidin (1000-fold dilution in PBS with 0.5% BSA) (Vector Laboratories Inc., Burlingame, CA, USA), respectively. The amount of HRP in each well was measured colorimetrically with the chromogenic reagent tetramethylbenzidine (Wako, Osaka, Japan). EC50 binding values were calculated with Prism 5 (GraphPad Software, La Jolla, CA, USA).
Maxisorp microplate
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Maxisorp microplates are a type of high-quality polystyrene microplate designed for use in various immunoassay applications. The Maxisorp surface provides a high-binding capacity for the immobilization of proteins, peptides, and other biological molecules. These microplates are suitable for a range of immunological techniques, including ELISA, cell-based assays, and other assays that require a consistent, reliable surface for capturing and detecting target analytes.
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Lab products found in correlation
Multiskan fc, by Thermo Fisher Scientific (4 mentions)
1 step ultra tmb blotting solution, by Thermo Fisher Scientific (3 mentions)
Polyethylene glycol 1500, by Merck Group (2 mentions)
Multiskan go, by Thermo Fisher Scientific (2 mentions)
Tetramethylbenzidine, by Fujifilm (1 mentions)
Hrp conjugated streptavidin, by Vector Laboratories (1 mentions)
Prism 5, by GraphPad (1 mentions)
Anti m13 hrp mab, by GE Healthcare (1 mentions)
Tetramethylbenzidine substrate, by Cell Signaling Technology (1 mentions)
Tmb liquid substrate, by Merck Group (1 mentions)
Hrp anti m13 monoclonal conjugate, by GE Healthcare (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Sulfuric acid, by Merck Group (1 mentions)
Mrx 2, by Dynex (1 mentions)
Horseradish peroxidase hrp conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
1 step ultra tmb elisa substrate, by Thermo Fisher Scientific (1 mentions)
Powerwave xs microplate reader, by Agilent Technologies (1 mentions)
Rhe selectin fc chimera, by R&D Systems (1 mentions)
1 step ultra tmb solution, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Bio-Rad (1 mentions)
26 protocols using maxisorp microplate
1
Fc-Fusion Protein Binding Assay
2
Quantifying Bacterial Surface Pili
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PilS detection was performed as previously described [20 (link)]. Briefly, bacterial cells were grown overnight in LB at 37 °C and then incubated in Dulbecco’s Modified Eagle Media (DMEM) containing 2% glucose in a 96-well MaxiSorp microplate (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) in a 1:50 dilution. After 4 h of growth, the medium was removed and non-adherent bacteria were removed by five washes with phosphate-buffered saline 0.01 M pH 7.2 (PBS). Cells were fixed for 15 min with PBS containing 2% glutaraldehyde and 3 mM CaCl2 followed by a single washing with PBS. Next procedure is the blocking step with PBS containing 1% bovine serum albumin (BSA) for 16–18 h at 4 °C in a humid chamber. The washing solution used from the blocking step between the subsequent steps was PBS—Tween 0.05% for 3 times.
After the blocking step, the cells were incubated for 1 h at 37 °C with an anti-PilS polyclonal antibody (1:500) followed by goat anti-mouse IgG peroxidase conjugate (1:5000) for 30 min at 37 °C. The reaction was developed using 0.5 mg/mL O-phenylenediamine (OPD; Sigma Aldrich Co., Saint Louis, MO, USA) in addition to 0.5-µL/mL hydrogen peroxide in 0.05 M citrate-phosphate buffer, in the dark at room temperature. The absorbance was measured at wavelength at 492 nm in a Multiskan FC (Thermo Fisher Scientific, Waltham, MA, USA).
After the blocking step, the cells were incubated for 1 h at 37 °C with an anti-PilS polyclonal antibody (1:500) followed by goat anti-mouse IgG peroxidase conjugate (1:5000) for 30 min at 37 °C. The reaction was developed using 0.5 mg/mL O-phenylenediamine (OPD; Sigma Aldrich Co., Saint Louis, MO, USA) in addition to 0.5-µL/mL hydrogen peroxide in 0.05 M citrate-phosphate buffer, in the dark at room temperature. The absorbance was measured at wavelength at 492 nm in a Multiskan FC (Thermo Fisher Scientific, Waltham, MA, USA).
3
Candida albicans Binding Assay
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Aliquots of 5 × 105C. albicans cells per well of a MaxiSorp microplate (Nunc) were grown in 100 µL of RPMI 1640 medium at 37 °C for 18 h. The cells were then washed three times with 200 µL of PBS and incubated with (i) 50 µL of 0.1 µM PPAD with 0.015 nM HRgpA and Kgp for 2 h at 37 °C or (ii) 100 µL of media collected after growth of P. gingivalis strains ATCC 33277, ∆ppad (ATCC 33277), W83, Δppad (W83) for the next 18 h at 37 °C. After washing out of the bacterial enzymes with PBS, the reactions in the wells were blocked using 300 µL of 3% bovine serum albumin (BSA) for 1 h at 37 °C. Then, 50 µL of each human protein solution in the concentration range from 3.125 nM to 200 nM were added to C. albicans cells for 1.5 h at 37 °C. Bound proteins were detected with the SA-HRP/TMB (streptavidin conjugated to horseradish peroxidase/3,3′,5,5′-tetramethylbenzidine) detection system and absorbance at 450 nm was measured with a Power Wave X Select microplate reader (BioTek Instruments, Winoosky, VT, USA). This experiment was performed in three independent replicates.
4
Enzyme-Linked Immunosorbent Assay Protocol
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The ELISA was performed by immobilizing 2 μg/ml of the immunogen onto the Maxi-sorp microplate (Nunc, Thermo Scientific) in 0.1 M NaHCO3 (pH 8.9) buffer and then blocking with a PBS/0.2% BSA solution. After washing four times with PBS containing 0.05% Tween-20 (pH 7.2), the plate was then probed with the corresponding phages or antibodies and developed with the tetramethylbenzidine substrate (Cell Signaling Technology). The absorbance of the wells was measured on a plate reader (Tecan). For the phage ELISA, an anti–M13-HRP mAb (GE Healthcare Life Science, 1:5000 v/v) was used. For other ELISAs, a goat anti-human IgG HRP (BioLegend, 1:5000 v/v) was used [22] (link). The data were processed using Graphpad Prism 5.
5
Nanobody Selection against Shiga Toxin 2A
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Selection of Nbs against rStx2aB was performed with 100 µL of rStx2aB (100 µg/mL) immobilized in a well of a Maxisorp microplate (Nunc, Thermo Fisher Scientific, Waltham, MA, USA). After the second and third round of panning, single colonies were picked, cultured and induced with 1 mM IPTG to express the cloned Nb in the periplasm. Subsequently, the recombinant Nbs extracted from the periplasm (PE) were tested in an ELISA (PE-ELISA), to identify those clones that produce a Nb recognizing the rStx2aB antigen. The clones giving a positive signal in PE-ELISA were sent for sequencing and their predicted amino acid sequence was analyzed in silico and classified in families according to the amino acid diversity within the CDRs.
6
Capture and Detection of CD1b and CD1e Complexes
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A 96-well Maxisorp microplate (Nunc, United States) was coated (16 h/4°C) with: (a) the anti-CD1b mAb BCD1b3.1 (Sigma, United States) to capture CD1b complexes and free CD1b; (b) the anti-CD1e mAb CD1e20.6 to capture the recombinant human CD1e. Both mAbs were diluted in carbonate-bicarbonate pH 9.6 coating buffer (3 μg/mL). The plate was then blocked with 3% PBS-skim milk (1 h/RT). Two washes (PBS-tween-20, 0.05%) were performed, and the complexes and free CD1b were added in PBS pH7.4 (1 μg/mL). After three washes (PBS-tween20, 0.05%), scTCR and dAbκ11, diluted in 3% PBS-skim milk (108 phages) were added and the plate was incubated for 1 h at RT. Subsequently, three washes were performed, and HRP/Anti-M13 monoclonal conjugate (GE, Healthcare, Life Sciences; diluted 1:5000 in 3% PBS-skim milk) was added. 1 h later, the plate was washed as above, TMB liquid substrate (Sigma, United States) was added, followed by incubation (10 min/RT). The reaction was stopped with 1 M sulfuric acid (Merck, Germany). Absorbance values (450 nm) were measured in a microplate reader (BioRad, United States). Each sample was studied in triplicate, and the experiment was repeated three times.
7
Evaluating Antigenicity of PQ10 Protein via ELISA
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ELISA procedures were done to evaluate the antigenicity of multiepitope PQ10 protein. Maxisorp microplate (Nunc, Denmark) was coated overnight with 16 μg/ml PQ10 protein diluted in 0.1 M carbonate buffer (pH 9.6) at 4 °C. After three washes with PBS-0.05% Tween-20 (PBST) (PBS: 10.14 mM Na2HPO4; 1.37 mMKH2PO4; 146 mM NaCl; 2.64 mM KCl, pH 7.4, containing 0.05%Tween20), wells were blocked with 200 μl/well of 1% Bovine Serum Albumin (BSA) in PBS at 37 °C for 2 h. Serum samples, diluted 1:100 in PBS-0.05% Tween-20 containing 0.5% BSA, were added and incubated at 37 °C for 1 h. After three washes, microplate was incubated with anti-human IgG-HRP (from goat) (Sigma, reference A8667), diluted 1:30000 in PBS at 37 °C for 1 h. After washing five times, reactions were developed with Tetra Methyl Benzidine (TMB) (Biolegend, reference 421101) and the microplates were incubated for 20 min in the dark room. Reactions were stopped with 2M H2SO4, and microplate was read at 450 nm in a DYNEX (MRX II) ELISA reader.
8
Quantification of ΔLNGFR on Lentiviral Particles
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A flat-bottom, 96-well MAXISORP microplate (Thermo Fisher Scientific, Darmstadt, Germany) was coated with 0.1 μL of different LV particles in 100 μL PBS-T (PBS and 0.05% Tween-20). After blocking the wells with PBS-B (PBS and 0.5% BSA), the biotinylated anti-LNGFR antibody (clone ME20.4-1.H4, Miltenyi Biotec, Bergisch Gladbach, Germany) was added. To quantify the amount of ΔLNGFR on the vector particles, wells were incubated with horseradish peroxidase (HRP)-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Ely, UK) diluted 1:500 in PBS-T (PBS and 1% Tween-20). Thereafter, 1-Step Ultra TMB-ELISA substrate (Thermo Fisher Scientific, Darmstadt, Germany) was used to develop the color reaction. At the end, 1 N H2SO4 solution was added to stop the reaction, and measurement was performed at 450 nm and 630 nm by an ELISA reader. A serial dilution from 0 to 12.5 ng human recombinant NGFR (Sino Biological, Wayne, PA, USA) was used to draw standard curve. All protein or antibody incubation steps were performed for 1 h at room temperature, followed by washing the wells three times with PBS-T. Each experiment was repeated up to nine times with technical duplicates.
9
Sensitive a-PD-1 ELISA Sandwich Assay
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Figure 2 A shows the schematic representation of the developed a-PD-1 ELISA sandwich method. Briefly, 24 h before performing the assay, a flat-bottomed 96-well Maxisorp microplate (NUNC, Thermoscientific, Rochester, NY, USA) was coated with 2 μg/mL of recombinant mouse-PD-1 capture fusion protein diluted in DPBS and incubated at 4°C overnight. Afterward, the plate, washed twice with 200 μL/well of DPBS, was blocked with 200 μL/well of IB for 1 h at room temperature (RT) to prevent unspecific reactions. Then, the plate was washed five times using 200 µL/well of washing buffer (0.05% Tween-20, DPBS; WB), and the standards and experimental samples (100 μL/well) were added and incubated for 2 h at RT. For the labeling, the plate was washed and treated for 1 h with a secondary goat a-rat IgG antibody (1:20.000, v/v. IB). In order to amplify this signal, a-Goat IgG HRP-conjugated antibody was added (1:5.000, v/v. IB) and incubated for 1 h at RT.
The plate was washed and revealed with 100 μL/well of TMB for 3 min. This reaction was stopped by adding 50 μL/well of sulphuric acid (2 N), and the absorbance was read at 450 nm in PowerWave™ XS Microplate Reader from BioTek Instruments, Inc (Winooski, VT, USA).
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The plate was washed and revealed with 100 μL/well of TMB for 3 min. This reaction was stopped by adding 50 μL/well of sulphuric acid (2 N), and the absorbance was read at 450 nm in PowerWave™ XS Microplate Reader from BioTek Instruments, Inc (Winooski, VT, USA).
10
Quantifying E-selectin Cell Adhesion
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Cell adhesion to recombinant human E-selectin (rhE-selectin) was performed with minor modifications, as previously described [15 (link)], using 2 µg/mL of rhE-selectin/Fc Chimera (R&D Systems, Minneapolis, MN, USA), which was incubated to a Fc antibody (Millipore, Darmstadt, Germany) and was previously bound to a 96-well maxisorp microplate (Thermo Fisher Scientific, Waltham, MA, USA). A total of 75,000 cells/well for BxPC-3 and 50,000 cells/well for Capan-1, treated and non-treated with Ac53FaxNeu5Ac, were seeded by quintuplicate into the rhE-selectin coated plates and incubated for 1 h at 37 °C. After washings, the remaining rhE-selectin-bound cells were estimated with the cell proliferation assay MTS (Promega, Madison, WI, USA), following the manufacturer’s protocol. The mean absorbance of the adhered cells was normalized by the corresponding negative control (wells without rhE-selectin). Cell adhesion was expressed by dividing the mean absorbance of treated cells by the mean of the untreated cells.
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